Carbohydrate Research, 238 (1993) 287-306 Elsevier Science Publishers B.V.. Amsterdam 287 Facile enzymic de mm synthesis and NMR spectroscopic characterization of D-tagatose 1,6-bisphosphate * Oliver Eyrisch, Gudrun Sinerius and Wolf-Dieter Fessner fiprtnzent of Org&c C&rtristry and ~~~~f~, Univemity of Freiburg, Albertstr. 22, D-7800 Fre&wg ~~e~9~ (Received February 2Oth, 1992; accepted June lst, 1992) ABSTRACC A D-tagatose l,~b~ph~phate aldolasc requiring Zn ‘+ for catalytic activity (class II) was purified from E. co& c&s grown on galactitol. The aldolase, a homotetramer composed of subunits of mol wt _ 28000, had a pH optimum at 7.5 and was highly selective for t-erythro as compared to D-thmo stereochemistry (99: 1). This allowed its application in a coupled enzyme system together with glycerol kinase, pyruvate kinase and triose phosphate isomerase for the de nova, one-pot synthesis of D-tagatose 1,6-bisphosphate starting from dihydroxyacetone and ph~pb~noi~vate ffor the in situ regeneration of adenosine t~phosphatc), in quantities of 10 mmol. The expeditious process compares very favorably in simplicity and yield (40% overall) with the known multistep chemical preparation even after improvements to the latter accomplished during the present work. The classical sequence, which starts from o-galacturonic acid, was modified at both phosphorylation steps: 1,2:3,4-di-O-isopropylidene-n- tagatofuranose was esterified by application of the trivalent phosphitylation agent dibenzyl di-N-ethyl- phosphoramiditc followed by hydrogen peroxide oxidation, and a bacterial fructose 6-phosphate kinase was used for enzymic ph~ph~lation of o-tagatose t&phosphate. For the latter enzyme, which was the isoenzyme Pfk-2 from a ~~rn~ant strain of E. coli, kinetic constants were determined. NMR spectroscopic assignments are presented for o-tagatose and its phosphates. INTRODUCX’ION Sugar phosphates play an important physioio~cal role as ~te~ediates and regulators in carbohydrate metabolism’. While their chemical synthesis constitutes a considerable challenge2, enzymic methods provide an appropriate and attractive alternative3. An increasing number of aldolases are being introduced as valuable catalysts for asymmetric synthesis4, with a notable emphasis on applications towards sugar$. Particularly, the use of a set of four ste~~hemic~~y implemental microbial aldolases has been proposed for the directed de nouo synthesis of monosaecha- Cumspo&vzce to: Dr. W.-D. Fessner, Department of Organic Chemistry and Biochemistry, University of Freiburg, Albertstr. 21, D-7800 Freiburg, Germany. * Enzymes in Organic Synthesis, Part 4. For Part 3, see ref. 8. 0008-6225/93/$06.00 8 1993 - Elsevier Science Publishers B.V. All rights reserved